One of the main challenges facing today's tomato industry is how to deliver to a processing plant or to the marketplace tomato fruit that have been vine-ripened (and thus are desirable to consumers in terms of taste, texture, and color), but that remain firm without the usual post-harvest ripening-related softening that reduces shelf life of harvested fruit. Using traditional breeding methods, which are very labor intensive, it could take years to develop a novel tomato variety that ultimately may display only a modest increase in shelf life. Instead, recent studies have utilized genetic and biochemical techniques in an effort to identify the factors that regulate fruit ripening. By identifying and modifying the expression of specific genes, researchers and breeders hope to develop new tomato varieties that have the desirable qualities of vine-ripened fruit, but that are resistant to post-harvest softening and therefore display an extended shelf life.
Ripening is a complex process involving numerous physiological and biochemical changes including changes in color, firmness, sugar content, and pathogen resistance. Post-harvest ripening limits the shelf life of fresh produce, such as tomatoes. Several genes involved in the ripening process have been identified by analysis of single locus mutations that result in a non-ripening phenotype. One of these genes has been called NOR after a naturally occurring mutation at the nor (non-ripening) locus of tomato. The non-ripening phenotype results from a 2 base pair deletion in the NOR gene, which causes a frame shift that affects NOR protein synthesis (see U.S. Pat. No. 6,762,347). This NOR deletion mutation severely impairs tomato fruit ripening and causes a broad range of undesirable traits that have proven difficult to eliminate through traditional breeding. It is the only characterized mutation in the NOR gene of tomato. NOR is a member of the NAC protein family, a large family of plant-specific transcription factors involved in multiple developmental processes, including formation of shoot apical meristem, floral organs, lateral shoots, hormone control and defense mechanisms, and programmed cell death. The structure of the DNA-binding NAC domain has recently been determined (Ernst et al., EMBO Reports 5(3):297-303, 2004; Olsen et al., Trends in Plant Science, 10(2):79-97, 2005).
Standard breeding methods have utilized the NOR deletion mutation in tomatoes (see U.S. Pat. No. 6,180,854). The usefulness of this deletion mutation is limited however since these mutant fruit fail to ripen normally. While the NOR deletion mutant fruit may have an increased shelf life, they have decreased sensory qualities (e.g., impaired flavor, aroma, and color) compared to wild type fruit which makes the nor deletion mutant fruit less appealing to consumers. Fruit that are homozygous for the NOR deletion mutation fail to ripen and remain hard and green. Breeders have attempted to use the NOR deletion mutation in the heterozygous state to develop firmer fruit. Even fruit that are heterozygous for the NOR deletion mutation fail to fully develop the red color and sensory qualities that consumers desire in ripened fruit.
To date, other useful characterized mutations in the NOR gene of tomato are not available. Because NOR exerts pleiotropic effects, it would be useful to have an allelic series of mutations in the NOR gene that provide a spectrum of firmness and color phenotypes that could be used to optimize the breeding of extended shelf life tomato varieties that retain many of the quality traits of vine-ripened tomatoes. Additional useful NOR mutations would include those that increase shelf life but do not affect flavor, aroma and color as adversely as the naturally occurring nor deletion mutation. Tomato lines with NOR mutations that have been genetically characterized could also be crossed with lines that carry mutations in other genes involved in ripening.
In addition to standard breeding methods utilizing the nor mutation, transgenic approaches that targeted the NOR gene (see U.S. Pat. No. 6,762,347; U.S. Patent Application No. 20050076410) have been proposed for tomato fruit development. However, public acceptance of genetically modified plants, particularly with respect to plants used for food, is not universal. Because a cultivated tomato that is resistant to post-harvest softening and has improved shelf life with quality traits acceptable to consumers would be useful, an allelic series of novel mutations in the NOR gene of tomato were created. A cultivated tomato with reduced fruit softening as a result of altered NOR that was not the result of genetic engineering would have tremendous value for the tomato industry, including fresh market tomatoes, processor tomatoes and tomato food products such as sliced tomatoes, canned tomatoes, ketchups, soups, sauces, juices and pastes.